Possible Effects of Glyphosate on Mucorales Abundance in the Rumen of Dairy Cows in Germany

Glyphosate (N-phosphonomethyl glycine) is registered as a herbicide for many food and non-food crops, as well as non-crop areas where total vegetation control is desired. Glyphosate influences the soil mycobiota; however, the possible effect of glyphosate residues in animal feed (soybean, corn, etc.) on animal mycobiota is almost unknown. Accordingly, the present study was initiated to investigate the mycological characteristics of dairy cows in relationship to glyphosate concentrations in urine. A total of 258 dairy cows on 14 dairy farms in Germany were examined. Glyphosate was detected in urine using ELISA. The fungal profile was analyzed in rumen fluid samples using conventional microbiological culture techniques and differentiated by MALDI-TOF mass spectrometry. LPS-binding protein (LBP) and antibodies (IgG1, IgG2, IgA, and IgM) against fungi were determined in blood using ELISA. Different populations of Lichtheimia corymbifera, Lichtheimia ramosa, Mucor, and Rhizopus were detected. L. corymbifera and L. ramosa were significantly more abundant in animals containing high glyphosate (>40 ng/ml) concentrations in urine. There were no significant changes in IgG1 and IgG2 antibodies toward isolated fungi that were related to glyphosate concentration in urine; however, IgA antibodies against L. corymbifera and L. ramosa were significantly lower in the higher glyphosate groups. Moreover, a negative correlation between IgM antibodies against L. corymbifera, L. ramosa, and Rhizopus relative to glyphosate concentration in urine was observed. LBP also was significantly decreased in animals with higher concentrations of glyphosate in their urine. In conclusion, glyphosate appears to modulate the fungal community. The reduction of IgM antibodies and LBP indicates an influence on the innate immune system of animals.     

Polysome assembly and RNA synthesis during phytochrome-mediated photomorphogenesis in mustard cotyledons

The effect of phytochrome (high irradiance reaction, elicited by continuous far-red light) on cellular polysome levels was investigated using ribosome-isolation procedures which prevent the methodological artifacts inherent in previous studies on polysomes. By including the large pool of ribosomal subunits in the analysis and using the ratio (polysomes: monomers + subunits) as a quantitative estimate of the translational capacity of the ribosomes in mustard (Sinapis alba L.) cotyledons, we found the following results: 1) After a lag-phase of less than 30 min, phytochrome induces a massive increase in the relative amount of cytosolic (free) polysomes at the expense of ribosomal subunits. 2) Cytosolic and membrane-bound polysomes are increased by phytochrome in constant proportions (constant ratio of 65:35 in light and darkness). 3) Simultaneously with the light-mediated increase of the polysome level there is an increased incorporation of newly synthesized (labeled) non-ribosomal RNA, presumably mRNA, into the polysomes which can be kinetically discriminated from the slower incorporation of newly synthesized (labeled) rRNA. 4) Cordycepin strongly inhibits the synthesis of RNA and completely prevents the light-mediated increase of polysomes. 5) The electrophoretic patterns of the in-vitro translation products obtained with polysomal polyadenylated RNA from dark-grown and light-grown cotyledons showed no significant qualitative differences. We conclude from these results that photomorphogenesis of mustard cotyledons is related to a massive increase of newly synthesized mRNA leading to a correspondingly increased recruitment of ribosomal subunits into polysomes. The phytochrome-induced increase of translatable mRNA involves mainly quantitative changes in the production of mRNA species which are also present in the dark-grown cotyledons.     

Production of IgM monoclonal antibodies in DG44 cells

Two-bicistronic vectors for the production of recombinant IgM monoclonal antibodies in the DG44 DHFR-negative cell line have been designed. We used tandem vectors, in which one bicistronic unit encoded the immunoglobulin light chain and DHFR and the other encoded the heavy chain and EGFP. The construct structure presumes that green cells surviving selection would be capable of producing both immunoglobulin chains. We found that the agglutinating IgM antibodies could be secreted in the absence of J-peptide. It was shown that the germinal leader peptide plays a key role in the expression of the genes for the light and heavy chains. A comparison of the chromatin regulatory elements demonstrated that construct-flanking 2xHS4 insulators stabilized the biosynthesis of the recombinant antibodies, whereas the 5′-MARLyz matrix attachment region proved to be less efficient. The strategy for obtaining a DG44-based producer cell line should include the following consecutive steps: selection on the medium without nucleoside → amplification of the inserted gene → cloning of transfectants → selection of high-productive clones. An attempt to clone before amplification and to amplify individual clones failed to result in effective producers. Cloning on a medium without selection pressure allows a more adequate assessment of the stability of the antibody production.     

Production of plant growth substances by rhizosphere mycoflora of broad bean and cotton

The culture filtrates of the rhizosphere fungi of broad bean (Vicia faba L.) and cotton (Gossypium barbadense L.) were analysed for the presence of plant growth substances of auxin and gibberellins nature. Bioassay test and chromatographic analysis indicated that these fungi, each synthesized different auxins in their culture medium. These auxins were indole compounds. Similarly the rhizosphere fungi produced in their culture medium some gibberellins and gibberellin-like substances.     

Up-regulation of MDP and tuftsin gene expression in Th1 and Th17 cells as an adjuvant for an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus

The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.     

Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to pisum and Avena phytochrome

Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome—red-absorbing form and phytochrome—far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action.     

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: Codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1–6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F₁ hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1^b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1^a) mice were low responders. The V_H recombinant strains BAB.14 and CB-8KN that possess the Ig-1^b allotype of C57BL, but have some of theV _H genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.     

Chemical fingerprinting and antibacterial activity of Saussurea lappa clarke

Costunolide and dehydrocostus lactone of Saussurea lappa are the active compounds having various biological activities used in medicine. HPLC and HPTLC were used for chemical profiling of S. lappa samples collected from different geographical regions of Uttarakhand, India. Costunolide was found to be 0.19 to 0.39% and 0.25 to 0.56% while dehydrocostus lactone was reported in the ranges of 0.27 to 0.70 and 0.50 to 1.06% by HPLC and HPTLC, respectively. Antibacterial activity of methanol extracts of S. lappa was evaluated in order to compare the effects of active constituents against gram positive and negative bacteria. It was determined by well diffusion method. S. lappa exhibited inhibitory effect on bacterial strains with the MICs ranging from 3.12 to 12.50 μg/μL for Escherichia coli and Citrobacter freundii, from 6.25 to 25.00 μg/μL for Enterococcus faecalis and from 6.25 to 50.00 μg/μL for Staphylococcus aureus. It was observed that the samples of S. lappa containing the higher contents of costunolide and dehydrocostus lactone showed the higher antibacterial activity. The results demonstrated that concentrations of both active constituents depended on altitude at which the collected plants were located.     

Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.)

A synthetic gene encoding the antimicrobial peptide magainin has been designed, cloned, and engineered for regulation by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (nos) terminator. The plant expression cassette was introduced into the vector pSB11-bar (with the glyphosate [Basta®] resistance gene, bar), and the recombinant plasmid was mobilized into Agrobacterium tumefaciens strain LBA4404 for the generation of a super-binary vector pSB111-bar-mag. Magainins, positively charged amphipathic antimicrobial peptides of 21–26 amino acid residues, are potential candidates for the development of disease resistant transgenic plants. Six-wk-old pearl millet (Pennisetum glaucum [L.] R. Br.) calli and A. tumefaciens harboring pSB111-bar-mag were cocultivated in a medium supplemented with 400 μM acetosyringone and 3.3 mM l-cysteine. Out of 3,000 infected calli subjected to selection on phosphinothricin medium, 82 calli showed sectors of healthy growth, resulting in a transformation frequency of 2.73%. Among 13 Basta-tolerant putative transformed plants, eight were fertile and their transgenic nature and expression of the transgene was characterized by Southern and Northern blot analyses, respectively. Subsequent T₁ progenies co-segregated for bar and magainin genes in a 3:1 ratio. Bioassays that challenged the eight transgenic T₁ plant progenies against three highly virulent strains of Sclerospora graminicola, viz., Sg 384, Sg 445, and Sg 492 failed to show resistance. The failure of synthetic magainin gene to confer resistance against downy mildew in pearl millet may be attributed to the complexity of the cell wall and cell membrane of the pathogen.     

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes k ₁ for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients k ₁ probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/L) and DMSO (2250 mOsm/L) the filtration coefficient L _p in the presence of a penetrating cryoprotectant (L _pDMSO = (4.45 ± 0.04) · 10^?14 m³/Ns) is 3 orders lower compared to the case with electrolyte (L _pNaCl = (2.25 ± 0.25) · 10^?11 m³/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.     

The Development of Monoclonal Antibodies to the secA Protein of Cape St. Paul Wilt Disease Phytoplasma and Their Evaluation as a Diagnostic Tool

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.     

Population ecology of Sulfolobus acidocaldarius

Ecology of Sulfolobus acidocaldarius was studied in situ by the use of the immunofluorescence and immunodiffusion techniques. The fluorescent antibodies (FA) prepared against four strains of Sulfolobus were highly reactive against their homologous antigens. Two of the FA's were strain specific and the other two exhibited reciprocal corssreactions against each other's antigens, but immunodiffusion patterns showed that the two strains were not identical. The growth of a serologically distinct isolate in a hot spring was measured by immunofluorescence staining of immersion slides. On glass immersion slides Sulfolobus grew and formed colonies with a mean-doubling time of approximately 36 h. Immunofluorescence was applied to study the geographical distribution of two serologically different strains and to establish population composition of individual springs. One strain was found in all sites studied, and most springs contained more than one serologic type. Immunodiffusion was capable of detecting specific Sulfolobus antigens in hot springs which contained a high population of FA-reactive cells.     

Influence of bovine lactoferrin on the growth of selected probiotic bacteria under aerobic conditions

Bovine lactoferrin (bLf) is a natural glycoprotein, and it shows broad-spectrum antimicrobial activity. However, reports on the influences of bLf on probiotic bacteria have been mixed. We examined the effects of apo-bLf (between 0.25 and 128 mg/mL) on both aerobic and anaerobic cultures of probiotics. We found that bLf had similar effects on the growth of probiotics under aerobic or anaerobic conditions, and that it actively and significantly (at concentrations of >0.25 mg/mL) retarded the growth rate of Bifidobacterium bifidum (ATCC 29521), B. longum (ATCC 15707), B. lactis (BCRC 17394), B. infantis (ATCC 15697), Lactobacillus reuteri (ATCC 23272), L. rhamnosus (ATCC 53103), and L. coryniformis (ATCC 25602) in a dose-dependent manner. Otherwise, minimal inhibitory concentrations (MICs) were 128 or >128 mg/mL against B. bifidum, B. longum, B. lactis, L. reuteri, and L. rhamnosus (ATCC 53103). With regard to MICs, bLf showed at least four-fold lower inhibitory effect on probiotics than on pathogens. Intriguingly, bLf (>0.25 mg/mL) significantly enhanced the growth of Rhamnosus (ATCC 7469) and L. acidophilus (BCRC 14065) by approximately 40–200 %, during their late periods of growth. Supernatants produced from aerobic but not anaerobic cultures of L. acidophilus reduced the growth of Escherichia coli by about 20 %. Thus, bLf displayed a dose-dependent inhibitory effect on the growth of most probiotic strains under either aerobic or anaerobic conditions. An antibacterial supernatant prepared from the aerobic cultures may have significant practical use.     

Estimates of heavy metal tolerance and chromium(VI) reducing ability of Pseudomonas aeruginosa CCTCC AB93066: chromium(VI) toxicity and environmental parameters optimization

The potential role of parameters in the reduction of hexavalent chromium [Cr(VI)] by Pseudomonas aeruginosa is not well documented. In this study, laboratory batch studies were conducted to assess the effect of a variety of factors, e.g., carbon sources, salinity, initial Cr(VI) concentrations, co-existing ions and a metabolic inhibitor, on microbial Cr(VI) reduction to Cr(III) by P. aeruginosa AB93066. Strain AB93066 tolerated up to 400 mg/L of Cr(VI) in nutrient broth medium compared to only 150 mg/L of Cr(VI) in nutrient agar. This bacteria exhibited different levels of resistance against Pb(II) (200 mg/L), Cd(II) (100 mg/L), Ni(II) (100 mg/L), Cu(II) (100 mg/L), Co(II) (50 mg/L) and Hg(II) (5 mg/L). Cr(VI) reduction was significantly promoted by the addition of glucose and glycerine but was strongly inhibited by the presence of methanol and phenol. The rate of Cr(VI) reduction increased with increasing concentrations of Cr(VI) and then decreased at higher concentrations. The presence of Ni(II) stimulated Cr(VI) reduction, while Pb(II), Co(II) and Cd(II) had adverse impact on reduction ability of this strain. Cr(VI) reduction was also inhibited by high levels of NaCl, various concentrations of sodium azide and 20 mM of SO₄ ^2?, MoO₄ ^2?, NO₃ ^?, PO₄ ^3?. No significant relationship was observed between Cr(VI) reduction and redox potential of the culture medium. Scanning electron microscopy showed visible morphological changes in the cells due to chromate stress. Fourier transform infrared spectroscopy analysis revealed chromium species was likely to form complexes with certain functional groups such as carboxyl and amino groups on the surface of P. aeruginosa AB93066. Overall, above results are beneficial to the bioremediation of chromate-polluted industrial wastewaters.